The broad goal of this project is to contribute to elucidating the genetic basis of human disease. The immediate goals of the work are to define the biochemical basis of selective and competitive messenger RNA (mRNA) translation and to analyze the structure and function of specific ribonucleoprotein complexes. RNA-protein complexes have essential enzymatic and structural roles in many biological processes, including protein biosynthesis, virus replication, and virus assembly. The model system used in this project is a group of positive-sense single-stranded RNA viruses, alfalfa mosaic virus and ilarviruses, which are closely related to human alphaviruses that cause severe encephalitis. The regulation of viral coat protein mRNA translation and the specificity and relative simplicity of viral RNA-coat protein interactions in this model system offer important advantages for study. Three significant accomplishments of the current funding period are 1) the discovery of a novel RNA binding consensus sequence in the viral coat proteins, 2) the identification, through use of chemical modification interference and hydroxyl radical footprint analysis, of the coat protein binding site on viral RNA, and 3) the characterization of 3' untranslated sequences as key regulators of viral coat protein mRNA translational efficiency. The Specific Aims of the renewal proposal are to define precise RNA-protein contacts for the coat protein-RNA interaction and to elucidate the biochemical basis for the translational control mediated by the viral mRNA 3' untranslated region. One hypothesis to be tested is that a key arginine in the RNA binding consensus contacts guanine functional groups in the RNA. Several approaches will be used to map RNA-protein contacts. Chemically synthesized RNAs containing specific base or ribose functional group modifications will be analyzed in RNA binding experiments, including native gel electrophoresis and hydroxyl radical footprinting. Peptides containing amino acid substitutions at highly conserved positions will also be used in determining if bound coat protein has alpha-helical character or if aromatic amino acids intercalate in the RNA helices. Circular dichroism data suggest that a coat protein peptide may stabilize an RNA conformational change upon binding. A sensitive technique, transient electric birefringence, will be used to characterize the conformational change by determining interhelical angles. A genetic method, based on translational repression in E. coli, will be used to rapidly screen gain-of-function mutants to identify candidate RNA-protein contacts. We propose that the 3' untranslated region enhances mRNA translation and competitive activity by recruiting translational components. The Xenopus laevis oocyte and yeast translation systems are being used to map 3' UTR sequence and structural determinants that enable the coat protein mRNA to out-compete other mRNAs, a strategy used by viral RNAs to usurp the host translational apparatus. Several approaches for high resolution structural analysis are proposed, including NMR and X-ray crystallography of chimeric virus particles expressing surface RNA- binding peptides.